Part:BBa_K2165001:Design

Violacein D gene codon-optimized for S. Cerevisiae

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 622
  Illegal SapI.rc site found at 1003
  

Design Notes

The VioD developed by the University of Washington's iGEM team have been codon optimized for yeast in hopes to improve the efficiency of its translation.

Source

The CDS contained in this BioBrick was designed by running the VioD gene sent to the University of Washington by the [http://dueberlab.berkeley.edu/ Dueber Laboratory] at University of California-Berkley through IDT's Codon Optimization Tool for S. cerevisiae. A biobrick standard assembly prefix and suffix was added before the it was ordered as a geneblock through IDT.

Characterization

Figure 1: Constituatively active VioABCDE yeast in synthetic media, along with a positive control (regular yeast) and a negative control (no yeast)

Figure 2: A gel electrophoresis of four parts related to the University of Washington's project.

Though typical biobrick characeterization involves in-vitro data, this is unavailabe due to the lack of an available chassis containing the three enzymes necessary to produce the substrate for VioD (VioA, VioB, and VioE). Because of the nature of this biobrick, it is reasonable to look at information from other sources to see the expected results. A protein BLAST shows that the sequence in this biobrick codes for "VioD" found in "cloning vector pET15b-vioD" with 100% quarry cover. Figure 1 shows an example of Violacein using the plasmid this biobrick was based from. the sequence in this biobrick codes for "VioC" found in "cloning vector pET15b-vioC" with 100% quarry cover. An image displaying the violacein pathway can be found [here] (Lee 2013; Kim 2016). This composite part should produce VioD in the presence of galactose. Providing VioA, VioB, and VioE are in solution, this will produce a tealish color pigment. When VioC is also present, a violet will be produced (shown in figure 1).

While the diagnostic gel for VioC had interesting results (the gel showed plasmid backbone and a band, resembling that of the composit part BBa_K2165002), we produced a biobrick with the proper size banding utilizing our BBa_K2165001, BBa_K2165003. We conclude that there must have been experimental error, but didn't have enough time to do another cut and gel before the jamboree. Figure 2 shows this diagnostic gel. VioC (lane 3) should have had a band at 1171 base pairs when cut with EcoRI and PstI; however, the [VioC composite part] has the expected band at 1963 base pairs and an additional band below what is presumed to be the backbone.

Works Cited

Kim, S. et al. PubChem substance and compound databases. Nucleic Acids Research 44, (2016).

Lee, M. E., Aswani, A., Han, A. S., Tomlin, C. J. & Dueber, J. E. Expression-level optimization of a multi-enzyme pathway in the absence of a high-throughput assay. Nucleic Acids Res. 41, 10668–10678 (2013).